The transcriptional repressor, REST (RE1 Silencing Transcription Factor;also called NRSF), was discovered in 1995 as a regulator or the gene encoding the voltage-dependent sodium channel, a protein crucial for the propagation of action potentials in most nervous systems. In the last funding period, by developing new reagents and techniques, we showed that REST control extends much farther than select excitability proteins;it regulates a large suite of genes that together define the neuronal phenotype. Many of these genes encode signature proteins of neuroendocrine tumors and may be involved in other types of epithelial cancers as well. We also found that REST controls a family of microRNAs that promote the neuronal phenotype in unexpected ways. Finally, we showed that, by orchestrating different chromatin and DNA modifications, REST plays important roles in controlling neuronal gene expression in pluripotent embryonic stem cells, neural stem/progenitors, and terminally differentiated cells. In the next grant period, we will 1) use a novel purification method to test two models by which REST recruits different chromatin complexes in different cellular contexts, 2) exploit a new mouse line we have generated to test whether REST plays major roles in neural stem/progenitors in adult neurogenesis as it does during embryogenesis, and 3) use a new method we have developed to elucidate comprehensive target mRNAs of miRNAs to understand how combinations of miRNAs function to promote the neuronal phenotype. Our studies will contribute insights into fundamental questions of gene regulation, as well as mechanisms that distinguish pluripotent from terminally differentiated phenotypes, an issue of obvious importance to understanding the origins of neural tumorigenesis. PUBLIC HEALTH RELEVANCE: REST continues to sit at the cutting edge of our understanding of fundamental cellular processes as well as neurogenesis. In the last funding period, in collaboration with the Elledge lab (HMS), we showed that certain human epithelial cancers are due to mis-expression of neuronal genes, linked likely to the dysregulation of REST. Our proposed studies represent several different attacks on the fundamental question of how control of neuronal gene chromatin is linked to formation of neural tumors, through studies of REST in different cellular contexts, from pluripotent embryonic stem cells, to neural stem/progenitors, to terminally differentiated non-neuronal cells.